Study On The Protective Effect And Mechanism Of Mountain-cultivated Ginseng Glycopeptide(APMCG-1) On Insulin Resistance-induced Different Muscle Injury

Objective: The quality of ginseng is closely related to its growth conditions and harvesting years.Mountain-cultivated ginseng refers to ginseng that is artificially sown and naturally grows for 15 years or more,which has high medicinal value.Most studies currently focus on the development and application of ginsenoside,with few studies related to ginseng residues after extraction.This thesis aims to explore the protective mechanism of enzyme extract on insulin resistance-induced muscle injury by re-extracting the residue after ethanol extraction from mountain-cultivated ginseng using an enzyme-assisted method based on the concept of green development.Methods:(1)After ethanol extraction,different types of enzymes were used to reextract residue from mountain-cultivated ginseng.The components obtained were then screened for in vitro antioxidant activity.The active ingredient APMCG-1 was identified through composition analysis and structure identification of the screened components.(2)The study evaluated the effects of APMCG-1 on endoplasmic reticulum stress and apoptosis by simulating heart injury of H9c2 cells under insulin resistance induced by palmitic acid.The study also examined the effects of APMCG-1 on heart injury,oxidative stress,and inflammatory level of Tg(kdrl:EGFP)zebrafish by establishing an insulin resistance model.(3)The effects of APMCG-1 on the apoptosis level of C2C12 cells were evaluated by simulating skeletal muscle injury under insulin resistance induced by palmitic acid.The effects of APMCG-1 on skeletal muscle injury,oxidative stress and inflammation in zebrafish were evaluated by establishing an insulin resistance model.The effect of APMCG-1 on the disorder pathway of glucose and lipid metabolism in zebrafish skeletal muscle was analyzed by metabolomics study.(4)16S r DNA high-throughput sequencing technique was used to analyze the effect of APMCG-1 on the diversity of intestinal flora in patients with insulin resistance.Results:(1)The mountain-cultivated ginseng’s alkaline protease extract(APMCG)has a significant capacity to scavenge hydroxyl radicals,and APMCG-1,which was obtained through additional separation and screening,has the ability to protect H9c2 and C2C12 cells from palmitic acid-induced damage.Thin layer chromatography,Fourier transform infrared spectroscopy,ultraviolet spectroscopy,high performance liquid chromatography,gel permeation chromatography and gas chromatography-mass spectrometry were used to determine that APMCG-1 was a glycopeptide.(2)APMCG-1 has been shown to reduce endoplasmic reticulum stress proteins such as GRP78,p-PERK,p-e IF2α,ATF4,CHOP,and mitochondrial apoptosis proteins like Bax and Caspase3 through the PI3K/AKT pathway in H9c2 cells.This effect is observed in cells that have undergone palmitic acid-induced lipid accumulation.APMCG-1 has also been shown to control heart rate and blood flow velocity in insulin-resistant Tg(kdrl:EGFP)zebrafish.It reduces lipid buildup on blood vessel walls and enhances SOD,GSH-PX,and CAT while decreasing FBG,T-CHO,TG,CK-MB,c Tn I,TNF-α,IL-6,and MDA.Additionally,APMCG-1 can lessen structural disruption and apoptosis in the heart of AB insulin-resistant zebrafish.(3)APMCG-1 has been shown to decrease lipid accumulation in C2C12 cells by decreasing mitochondrial apoptotic proteins Bax,Caspase9,and Caspase3 via the IRS1/PI3K/AKT/GLUT4 pathway in response to palmitic acid stimulation.In AB insulinresistant zebrafish,APMCG-1 can decrease FBG,INS,T-CHO,TG,FFA,MDA,and TNF-αlevels while boosting MG,SOD,GSH-PX,and CAT levels.AKT protein expression is also improved while apoptosis is decreased.The metabolomics study shows that APMCG-1controls disordered glucose and lipid metabolism by regulating cysteine,methionine,arginine and proline metabolism as well as steroid hormone production.(4)The relative abundance of Megamonas,Escherichia＿Shigella,and Limosilactobacillus was markedly elevated in individuals with insulin resistance when treated with APMCG-1.Conclusion: APMCG-1,as a PI3K/AKT activator,protects oxidative-induced cardiomyocyte and skeletal myoblast dysfunction,reducing levels of apoptosis in the heart and muscle tissue of insulin-resistant zebrafish,while improving the relative abundance of gut bacteria.These results suggest that APMCG-1 may be a potential therapeutic agent for the treatment of muscle injury induced by insulin resistance.